RNA Transcription in Prokaryotes

CELL AND MOLECULAR BIOLOGY

Learning Outcomes
•Describe the function and significance of genes and gene expression
•Differentiate structural genes from non-structural genes
•Discuss the process of RNA transcription in prokaryotic cells
Gene
•At the molecular level, a gene is defined as a segment of DNA that is used to
make a functional product
• Either an RNA molecule or a polypeptide
•The information within a gene is accessed beginning with the process called
transcription
• The process of synthesizing RNA from a DNA sequence
Structural Genes
•Encode amino acid sequence of a polypeptide
•When a structural gene is transcribed, the first product is an RNA molecule
known as messenger RNA (mRNA)
Transcription:The Beginning
•The molecular understanding of gene transcription initially came from studies
involving bacteria and bacteriophages
•The first suggestion that RNA is derived from the transcription of DNA was
made by Elliot Volkin and Lazarus Astrachan in 1956
Transcription:The Beginning
•In 1960, Matthew Meselson and Francois Jacob found that proteins are
synthesized on ribosomes
•A year later, Jacob & Jacques Monod proposed that a certain type of RNA acts
as a genetic messenger to provide the info. for protein synthesis
Transcription:The Beginning
•They hypothesized that this RNA, which they called messenger RNA (mRNA) is
transcribed from the sequence within DNA
• This will then direct the synthesis of particular polypeptides
•In 1961, the proposal was confirmed by Sydney Brenner in collaboration with
Jacob and Meselson
Overview
•Transcription occurs in three stages:
• Initiation, elongation and termination
•These steps involve protein-DNA interactions in which proteins like RNA
polymerase interact with DNA sequences
•The initiation stage in the transcription process is a recognition step
• The sequence of bases within the promoter region is recognized by
transcription factors
• The specific binding of transcription factor to the promoter sequence
identifies the starting site for transcription
Promoter Region
Promoter Region
•The DNA sequence that promotes gene expression
•This sequence of bases directs the exact location for the initiation of RNA
transcription
•Most of the promoter region is located just ahead of or upstream from a site
where transcription of a gene actually begins
Promoter Region
Promoter Region
•By convention, the bases in a promoter sequence are numbered in relation to
the transcriptional site
•This site is the first base used as a template for RNA transcription and is
denoted +1
• The bases preceding this site are numbered in negative direction
• No base is numbered zero
• Most of the promoter region is labelled with negative numbers that describe
the number of bases preceding the beginning of transcription
Promoter Region
Promoter Region
•In many promoter found in E. coli, and similar species, two sequence elements
are important
•These are located at approximately the -35 and -10 sites in the promoter region
•The sequence in the top DNA strand at the -35 region is 5’ –TTGACA- 3’
•The one at the top DNA strand is 5’ – TATAAT – 3’
• The TATAAT sequence is called the Pribnow box after David Pribnow
Promoter Region
Promoter Region
•The sequences at the -35 and -10 sites can vary among different genes
•The most commonly occurring bases within a sequence element form the
consensus sequence
• This sequence is efficiently recognized by proteins that initiate transcription
Promoter Region
RNA Polymerase Holoenzyme
•The enzyme that catalyzes the synthesis of RNA is the RNA polymerase
•In E. coli, the core enzyme is composed of five subunits, 2αββ’ω
• Two alpha, one beta, one beta prime, one omega
•The association of 6th subunit, sigma (σ) with the core enzyme is referred to as
RNA polymerase holoenzyme
RNA Polymerase Holoenzyme
RNA Polymerase Holoenzyme
•The two alpha subunits are important in the proper assembly of the
holoenzyme & in the process of binding to DNA
•The β and β’ subunits are also needed for binding to the DNA & carry out
catalytic synthesis of RNA
•The omega subunit is important for the proper assembly of the core enzyme
RNA Polymerase Holoenzyme
•The holoenzyme is required to initiate transcription
•The primary role of sigma (σ) factor is to recognize the promoter
•Proteins such as sigma factor that influence the function of RNA polymerase are
types of transcription factors
Initiation
Initiation
•After RNA polymerase holoenzyme is assembled into its six subunits, it binds
loosely to the DNA and then slides along the DNA
•When the holoenzyme encounters a promoter sequence, sigma factor protein
contains a structure called a helix-turn-helix motif that can bind tightly to these
regions
Initiation
•Alpha helices within the protein fit into the major groove of the DNA double
helix and form hydrogen bonds with the bases
Initiation
•Alpha helices fit into the major groove of the double helix and form hydrogen
bonds with the bases
•H-bonding occurs between nucleotides in the -35 & -10 regions of the
promoter & amino acid chains in the helix-turn-helix structure of the σ factor
Initiation
Initiation
•Transcription is initiated when sigma factor within the holoenzyme has bound to
the promoter region to form the closed complex
•For transcription to begin, the double-stranded DNA must then unwound into
an open complex
• This unwinding first occurs at the TATAAT sequence in the -10 region
Initiation
Initiation
•A short strand of RNA is made within the open complex, and then sigma factor
is released from the core enzyme
•The release of sigma factor marks the transition to the elongation phase of
transcription
•The core enzyme may now slide down the DNA to synthesize a strand of RNA
Elongation
Elongation
•The RNA transcript is made during the elongation stage
•During the synthesis of the RNA transcript, RNA polymerase moves along the
DNA, causing it to unwind
• The DNA strand used as a template for RNA synthesis is called the template
or antisense strand
• The opposite DNA strand is the coding or sense strand
Elongation
Elongation
•RNA polymerase always connects nucleotides in the 5’ to 3’ direction
• During this process, RNA polymerase catalyzes the formation of a bond
between the 5’ phosphate group on the nucleotide and the 3’ –OH group on
the previous nucleotide
•The complementarity rule is similar to the AT/GC rule, except that uracil
substitutes for thymine in the RNA
Termination
•The end of RNA synthesis is referred to as termination
•Prior to termination, the hydrogen bonding between the DNA and RNA within
the open complex is of central importance in preventing dissociation of RNA
polymerase from the template strand
•Termination occurs when this short RNA-DNA hybrid region is forced to
separate, thereby releasing RNA polymerase and the newly made RNA
transcript
Termination
•For certain genes, an RNA-binding protein known as ρ factor (rho factor) is
responsible for terminating transcription
• This mechanism is called ρ-dependent termination
•For other genes, termination does not require the involvement of the ρ protein
• This mechanism is called ρ-independent termination
Rho-dependent Termination
•In ρ-dependent termination, the termination process requires two components
•First, a sequence upstream from the terminator called the rut site for rho
utilization site, acts as a recognition site for the binding of the rho protein
Rho-dependent Termination
Rho-dependent Termination
•The rho protein functions as a helicase, an enzyme that can separate RNA-DNA
hybrid regions
•After the rut site is synthesized, in the RNA, rho protein binds to the RNA and
moves in the direction of RNA polymerase
Rho-dependent Termination
Rho-dependent Termination
•The second component of rho-dependent termination is the site where
termination actually takes place
•At this termination site, the DNA encodes an RNA sequence containing several
GC pairs that form a stem-loop structure
Rho-dependent Termination
•RNA synthesis terminates several nucleotides beyond this stem-loop
•A stem-loop structure, also called a hairpin, can form due to complementary
sequences with the RNA
•This stem-loop forms almost immediately after the RNA sequence is
synthesized and quickly binds to RNA polymerase
• This binding results in a conformational change that causes RNA polymerase
to pause in its synthesis of RNA
Rho-dependent Termination
•The pause allows rho protein to catch up to the stem-loop, pass through it and
break the hydrogen bonds between the DNA & RNA within the open complex
•When this occurs, the completed RNA strand is separated from the DNA along
with RNA polymerase
Rho-dependent Termination
Rho-independent Termination
•A process that does not require the rho protein
•In this case, the terminator is composed of two adjacent nucleotide sequences
that function within the RNA
• One is a uracil rich sequence located at the 3’ end of the RNA
• The second sequence is adjacent to the uracil-rich sequence and promotes
the formation of a stem-loop structure
Rho-independent Termination
•The formation of the stem-loop causes the RNA
polymerase to pause its synthesis of RNA
•This pausing is stabilized by other proteins that bind to
RNA polymerase
• A protein called NusA, which is bound to RNA
polymerase promotes pausing at stem-loop sequence
Rho-independent Termination
Rho-independent Termination
•At the precise time RNA polymerase pauses, the uracil-rich sequence in the
RNA transcript is bound to the DNA template strand
• Note: the hydrogen binding of RNA to DNA keeps RNA polymerase clamped
onto the DNA
•However, the binding of this uracil-rich sequence to the DNA template strand is
relatively weak
• This causes the RNA transcript to spontaneously dissociate from the DNA
and cease further transcription
Rho-independent Termination
•Because the process does not require a protein (the rho protein) to physically
remove the RNA transcript from the DNA
• This is referred to as intrinsic termination
•In E. coli about half of the genes show intrinsic termination, and the other half
are terminated by the rho protein
Summary
•Gene is defined as a segment of DNA that is used to make a functional product
•The first suggestion that RNA is derived from the transcription of DNA was
made by Elliot Volkin and Lazarus Astrachan in 1956
•Transcription in prokaryotes occurs in three stages: initiation, elongation and
termination
•RNA polymerase always connects nucleotides in the 5’ to 3’ direction
•The complementarity rule is similar to the AT/GC rule, except that uracil
substitutes for thymine in the RNA
References
•Karp, G. (2010). Cell and Molecular Biology. Hoboken, New Jersey, United States of
America: John Wiley and Sons, Inc.
•Karp, G. (2014). Cell Biology (7th Ed.). John Wiley & Sons Singapore Pte. Ltd. NJ,
USA
•Lewis, R. (2015). Human Genetics: Concepts and Applications. McGraw-Hill
Education. New York, New York, United States of America